We have constructed human artificial chromosomes (HACs) by modifying two different yeast artificial chromosomes (YACs) containing human centromeric a satellite DNA with human telomere sequences and putative origins of replication. The resulting HACs (250 and 1000 kb) which contain a neomycin resistance gene (neo r)have been introduced into mammalian cell lines and analyzed for their chromosomal function. Initial studies using fluorescence in situ hybridization (FISH) indicated that the HACs delivered to CHO cells by spheroplast fusion were integrated into the CHO genome. However, centromeric function was suggested for the larger HAC by the presence of a constriction and centromere binding proteins at the site of integration, and by the association of this integrated HAC with the induction of aneuploidy over time. Subsequently, both HACs have been transferred to human cells by liposome-mediated transfection of gel-purified DNA. Extrachromosomal elements containing the introduced a satellite DNA have been identified in neor transfectants generated from each HAC in the absence of integration into the human genome. Preliminary stability assessment indicates that the 1000 kb HAC is stable in the absence of G418 selection through 45 generations. We are currently pursuing these stability studies further. In addition, we are assessing the integrity and structural nature of the HACs compared to the material which we originally introduced into the human cells. Projects underway include (1) the introduction of the HAC into ES cells to generate transgenic mice; (2) developing strategies to transfer the HAC back to yeast for propagation, analysis and comparison to the original HAC; and (3) developing strategies to incorporate potentially therapeutic genes into a functional HAC.